Low abundant protein absolute quantification method based on digital immunoassay technology

An immunoassay and absolute quantification technology, which is applied in the field of protein ultra-sensitive analysis, can solve problems such as inability to make absolute quantification, and achieve the effects of high accuracy, high throughput, and high detection sensitivity

Inactive Publication Date: 2019-03-26
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to: aim at the deficiencies in the prior art above, to propose a low-abundance protein absolute quantification method based

Method used

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  • Low abundant protein absolute quantification method based on digital immunoassay technology
  • Low abundant protein absolute quantification method based on digital immunoassay technology
  • Low abundant protein absolute quantification method based on digital immunoassay technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Absolute quantitative analysis of CEA (carcinoembryonic antigen, carcinoembryonic antigen) standard

[0052] Preparation of magnetic capture beads: pipette 100 μL of magnetic beads (2×10 9 pcs / mL) stock solution in a 1mL centrifuge tube, washed twice with an equal amount of 0.01M NaOH solution, washed three times with an equal amount of deionized water, and then added 50 mg / mL EDC (i.e. 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride) solution, incubate at room temperature for 30 minutes, remove the supernatant by magnetic separation, add 60 μL capture antibody solution (antibody mass is 120 μg), incubate at room temperature for 30 minutes , and then added 100 μL of PBS (phosphate buffer saline, phosphate buffered saline) buffer (pH=7.4) containing 0.1% tween20 to wash 4 times, and finally obtained capture magnetic beads, which were stored in a 4°C refrigerator for later use.

[0053] Preparation of detection particles: Pipette 100 μL of particle...

Embodiment 2

[0056] Example 2 Absolute Quantitative Analysis of Plasma CEA

[0057] The operating steps of embodiment 2 are the same as embodiment 1, only the CEA standard solution is replaced with diluted 10 5 times as many human plasma samples. The number of molecules of CEA in the plasma sample is equal to the number of detected particles, and the concentration of CEA can be calculated by combining the sample volume and Avogadro's constant. The results are shown in Table 1. Table 1 is a comparison table of the absolute quantitative analysis results of CEA in 10 plasma samples, the hospital test results and the standard addition analysis results. Among them, a is diluted 10 5 times the plasma, b is the whole plasma.

Embodiment 3

[0058] Example 3 The standard addition analysis of plasma CEA

[0059] Dilute #5 plasma sample by 10 5 times, take 5 parts of diluted plasma (100 μL each), add 0, 5, 10, 15, 20 μL of CEA standard solution with a concentration of 200 aM respectively, the final concentration of added CEA is 0, 10, 20, 30, 40 aM, Subsequent operation steps are the same as in Example 1, only the CEA standard solution needs to be replaced by the plasma sample added with CEA. Take the final concentration of CEA added in the plasma as the abscissa, and the number of detected particles as the ordinate, draw a standard curve (such as Figure 5 ). Figure 5 , the reverse extension line of the standard curve intersects the X-axis at a point, the abscissa of the intersection point is 37.3, diluted 10 5 Times the #5 plasma sample CEA concentration is 37.3aM. The concentration of CEA in #5 plasma stock solution was 0.57ng / mL. Under the same conditions, the standard addition curve of #10 plasma sample i...

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Abstract

The invention relates to a low abundant protein absolute quantification method based on a digital immunoassay technology. The method comprises the following steps: carrying out an immunoreaction on captured beads, target antigens and detected particles so as to form an immune complex, eluting the detected particles in the immune complex, collecting and concentrating the eluant, transferring the concentrated solution to a micro-fluidic particle counting chip, depositing and fixing the detected particles on the micro-fluidic particle counting chip after a solvent in the concentrated solution iscompletely volatilized, performing imaging recording on all the detected particles fixed on the micro-fluidic particle counting chip, and counting the quantity of the detected particles on the micro-fluidic particle counting chip, thereby obtaining the molecular number of the target protein. The method disclosed by the invention has the advantages that ultra-sensitive and absolute quantification analysis of disease-related proteins in clinical samples can be realized, any standard substance is not needed, a standard curve does not need to be drawn, and the method has the advantages of being wide in application range, high in detection sensitivity, high in analytical result accuracy, excellent in precision degree and high in throughput.

Description

technical field [0001] The invention relates to an absolute quantitative method for low-abundance proteins based on digital immune analysis technology, in particular to an ultrasensitive absolute quantitative analysis method for low-abundance proteins in clinical samples, belonging to the technical field of protein ultrasensitive analysis. Background technique [0002] It is known that the occurrence and development of diseases are closely related to the abnormal expression of proteins or the expression of specific proteins. Accurate determination of the content of disease-related proteins is of great significance in the prevention and control of infectious diseases, cancer screening and accurate diagnosis. At present, protein quantification methods mainly include relative quantification and absolute quantification. The former is a method based on a standard curve, which is widely used in analytical chemistry and clinical testing; the latter does not require protein standar...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 张清泉盖宏伟张雪冰
Owner XUZHOU NORMAL UNIVERSITY
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